Abstract
The extension of the PEVK segment of the giant elastic protein titin is a key event in the elastic response of striated muscle to passive stretch. PEVK behaves mechanically as an entropic spring and is thought to be a random coil. cDNA sequencing of human fetal skeletal PEVK reveals a modular motif with tandem repeats of modules averaging 28 residues and with superrepeats of seven modules. Conformational studies of bacterially expressed 53-kDa fragment (TP1) by circular dichroism suggest that this soluble protein contains substantial polyproline II (PPII) type left-handed helices. Urea and thermal titrations cause gradual and reversible decrease in PPII content. The absence of sharp melting in urea and thermal titrations suggests that there is no long range cooperativity among the PPII helices. Studies with solid phase and surface plasmon resonance assays indicate that TP1 interacts with actin and some but not all cloned nebulin fragments with high affinity. Interestingly, Ca(2+)/calmodulin and Ca(2+)/S100 abolish nebulin/PEVK interaction. We suggest that in aqueous solution, PEVK is an open and flexible chain of relatively stable structural folds of the polyproline II type. PEVK region of titin may be involved in interfilament association with thin filaments in a calcium/calmodulin-sensitive manner. This adhesion may modulate titin extensibility and elasticity.
Highlights
The monumental sequencing work of Labeit and Kolmerer [1] has revealed the complete domain organization of the giant elastic protein titin
Conformational studies of bacterially expressed 53-kDa fragment (TP1) by circular dichroism suggest that this soluble protein contains substantial polyproline II (PPII) type left-handed helices
Recent works on the elasticity of single titin molecules [6], single myofibrils [7], and single fibers [8, 9] revealed that, stretched modestly, sarcomere elasticity can be explained by the straightening of tandem Ig segment, followed by the extension of a permanently unfolded PEVK segment [10]
Summary
Cloning and Sequencing of Human Fetal Skeletal Muscle PEVK—A gt human fetal skeletal muscle library was screened by Western blot with a goat anti-rabbit skeletal muscle titin (goat 812) that has been absorbed with E. coli blots and supplemented with a mixture of monoclonal antibodies (RT10, -11, -13, and -15) [8]. ELISA Protein Binding Assays—Purified TP1 (220 g/ml in 20 mM sodium phosphate, 1 mM EDTA, 150 mM NaCl, pH 7.0) was absorbed onto microtiter plates overnight at 4 °C, washed once with TBS-T (10 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.05% (v/v) Tween 20), and blocked with 0.02% (w/v) bovine serum albumin in TBS-T for 1 h at 37 °C, followed by incubation with actin and nebulin fragments (NA3, NA4, NC17, ND8, ND66) in buffer I (10 mM imidazole, 150 mM KCl, 1 mM CaCl2, 2 mM MgCl2, pH 7.0) at a concentration ranging from 0.7 to 50 g/ml for 2 h at 37 °C. The split muscle fibers from rabbit psoas muscle in relaxing buffer were hand-
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